Sample collection and morphological identification of soil arthropods:
To access soil arthropod biodiversity, sampling was done in October and November 2015 (a post-monsoon season) from a relatively undisturbed land (220 × 70m2) near the vicinity of the host institution (30ᵒ39’N 76ᵒ43’E, Mohali, Punjab, India; Figure S1A). The study area was naturally divided into roughly 8 quadrants by plantations of poplar (Figure S1B). Five randomly selected quadrants were sampled by collecting leaf litter and pitfall traps. These two independent sampling methods were used in tandem, since pitfall traps is biased for surface-active taxa, whereas leaf litter method is biased towards less active taxa, resulting in a more comprehensive sampling of the resident species (Olson, 1991; Querner & Bruckner, 2010). Two parallel transects, each 30m long, were marked across each quadrant using a rope (Figure S1B). Each of these transects were marked at 10m and two alternative marked points were sampled for leaf litter, while pitfall traps were placed at the other two ends (Figure S1C). Samples from each type of collections were later combined. In total, 20 collections, each from leaf litter and pitfalls, were obtained.
Leaf litter was collected from an area of roughly 0.09m2 (Figure S1D), and immediately placed in a plastic bag. Additionally, each leaf litter sample was accompanied by a soil sample of an area 282cm3 immediately below the leaf litter (Sabu & Shiju, 2010). Samples were weighed so that each sample roughly had the same weight (500-600gm) and were then settled in a series of Tullgren funnel with 100W light source. The emerging arthropods were collected in a 50 ml beaker, with absolute ethanol, continuously for the next 4-6 days or until no arthropod samples emerged. Emerged arthropods were collected every 24 hours and 100 ml of fresh absolute ethanol was added to the collection beaker. The pitfall traps were settled on the ground by placing a 250ml beaker with 50ml of absolute ethanol (Figure S1E). Samples were collected every second day with a replacement of fresh ethanol.
Arthropods obtained from each of pitfall and leaf litter samples (20 samples each) were sorted individually. Detailed (dorsal and lateral) views of each individual arthropod obtained were photographed under a stereomicroscope (M205C, Leica Microsystems) with scale varying from 0.2-2mm. These were then sorted according to their morphology (into morphospecies) and provisionally identified till order level. A total of 3509 individuals were sampled, which were categorized into 390 distinct morphospecies. Several diversity indices were calculated with EstimateS v9.1.0 (Colwell, 2013) like ACE (Abundance coverage estimator; Chao, Hwang, Chen, & Kuo, 2000), Chao1 (Chao, 1984), ICE (Incidence coverage estimator), Chao2 and Jack 1 and 2 (Smith & van Belle, 1984).
For morphospecies which had more than three individuals, DNA was extracted from a single individual by either the HiPurATM insect DNA purification kit (HIMEDIA) or by using the Phenol-Chloroform-Isoamyalcohol (PCI) method. In PCI method, samples were crushed in 200µl lysis buffer containing 10mM each of Tris-HCL (pH 8.0), EDTA (pH 8.0) and NaCl. DNA was precipitated using isopropanol and dissolved in 1X TE (pH 8.0). For morphospecies which only had single individuals, a different nondestructive extraction protocol was used (Rowley et al., 2007). Whole individuals were incubated at 60°C in 100-400µl of Guanidinium thiocyanate (GuSCN) based extraction buffer (GuSCN, 0.1M, Tris-HCL, 0.2M EDTA with Triton x-100) for 1-4hr. Then individuals were removed for storage and the DNA remaining in the buffer was precipitated using isopropanol. Extracted DNA was quantified using the NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific) and PCR-suitability was accessed by running a PCR reaction using 28S primers (Table S2).
The morphospecies were barcoded using (Hebert, Cywinska, Ball, & Dewaard, 2003) the ~600bp of the mitochondrialCO1 gene (Table S2). 2-20ng/µl of extracted DNA was used in 20µl PCR reactions with an initial denaturation step at 95°C for 3 minutes, 39 cycles of denaturation (95°C, 45 seconds), annealing (51-56°C for 45 seconds), extension (72°C, 1 minute) and a final extension at 72°C for 10 minutes. PCR products were visualized on 1% agarose gels and then cleaned with Exonuclease I and Shrimp alkaline Phosphatase (New England Biolabs Inc.). PCR products were sequenced using BigDye® Terminator v3.1 cycle sequencing kit. Initially, only the forward strand was sequenced and if any base ambiguity was observed then the reverse strand was also sequenced.
CO1 sequences obtained were identified through the NCBI (Johnson et al., 2008) and BOLD databases (Ratnasingham & Hebert, 2007) by BLAST (last performed in August 2019). The best hit obtained was used to check the provisionally identified morphospecies. If both databases yielded the same hit then it was determined to have been identified. If they yielded different hits then the taxonomic identification was moved down to the level common in these two hits. These results were further cross-referenced with the photographic data to finally build up a repository of identified morphospecies with their taxonomic ranks (Table S3). Only unique morphospecies were included in further analysis after analyzing the CO1 sequences.